title: DXP synthase-catalyzed C-N bond formation: nitroso substrate specificity studies guide selective inhibitor design.
abstract: 1-Deoxy-D-xylulose 5-phosphate (DXP) synthase catalyzes the first step in the nonmammalian isoprenoid biosynthetic pathway to form DXP from pyruvate and D-glyceraldehyde 3-phosphate (D-GAP) in a thiamin diphosphate-dependent manner. Its unique structure and mechanism distinguish DXP synthase from its homologues and suggest that it should be pursued as an anti-infective drug target. However, few reports describe any development of selective inhibitors of this enzyme. Here, we reveal that DXP synthase catalyzes C-N bond formation and exploit aromatic nitroso substrates as active site probes. Substrate specificity studies reveal a high affinity of DXP synthase for aromatic nitroso substrates compared to the related ThDP-dependent enzyme pyruvate dehydrogenase (PDH). Results from inhibition and mutagenesis studies indicate that nitroso substrates bind to E. coli DXP synthase in a manner distinct from that of D-GAP. Our results suggest that the incorporation of aryl acceptor substrate mimics into unnatural bisubstrate analogues will impart selectivity to DXP synthase inhibitors. As a proof of concept, we show selective inhibition of DXP synthase by benzylacetylphosphonate (BnAP).
authors.affiliation: Department of Pharmacology and Molecular Sciences, The Johns Hopkins University School of Medicine, 725 North Wolfe St, Baltimore, MD 21205, USA.
authors.forename: Francine
authors.initials: F
authors.lastname: Morris
authors.forename: Ryan
authors.initials: R
authors.lastname: Vierling
authors.forename: Lauren
authors.initials: L
authors.lastname: Boucher
authors.forename: Jürgen
authors.initials: J
authors.lastname: Bosch
authors.forename: Caren L
authors.initials: CL
authors.lastname: Freel Meyers
mesh_terms: D055162:Biocatalysis; D002244:Carbon / Q000737:chemistry; D020134:Catalytic Domain; D004791:Enzyme Inhibitors / Q000737:chemistry* / Q000378:metabolism; D007700:Kinetics; D009584:Nitrogen / Q000737:chemistry; D013379:Substrate Specificity; D013729:Terpenes / Q000737:chemistry / Q000378:metabolism; D014166:Transferases / Q000737:chemistry / Q000378:metabolism*
chemical_list: D004791:Enzyme Inhibitors; D013729:Terpenes; D002244:Carbon; D014166:Transferases; C109461:deoxyxylulose-5-phosphate synthase; D009584:Nitrogen
keywords: DXP synthase; biosynthesis; enzyme inhibitors; isoprenoids; kinetics; substrate specificity